Expression of protein kinase C ?1 confers resistance to TNF?- and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells

Abstract

The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor ␣ (TNF␣) in a dose-and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Key words: protein kinase C; TNF␣; paclitaxel; apoptosis; colon carcinomaColorectal cancer is one of the most common solid tumors world-wide. Due to its high metastatic potential and the frequent onset of resistance to chemotherapy, it is one of the four major causes of death by neoplasia in westernized countries. Loss of function of p53 occurs in more than 75% of human colorectal cancers. Since p53 regulates a complex array of cellular responses to DNA damage, including cell cycle arrest and apoptosis, its loss of function is also expected to affect the sensitivity of tumor cells to DNA-damaging antiblastic drugs. 1,2The expression of different protein kinase C (PKC) isoenzymes varies with proliferation rates, differentiation or apoptosis in normal colon crypts, [3][4][5][6] and the activity of some PKC isoenzymes is reduced in colorectal cancer. [7][8][9] Whether, in addition to loss of function of p53, altered functioning of PKCs contributes to the low sensitivity of colon carcinomas to apoptosis-based chemotherapy remains to be established. In the present work we addressed this issue by examining the effect of various apoptotic treatments on HT-29 colon cancer cells that lack functional p53 10 and either do or do not express abnormal levels of the isoform ␤1 of PKC. We found that paclitaxel (Taxol) and TNF␣, but not etoposide, efficiently induced apoptosis of HT-29 cells in a dose-and timedependent manner.The involvement of PKC in the apoptotic pathways triggered by paclitaxel or TNF␣ was assessed by using a myristoylated pseudosubstrate as inhibitor at doses not affecting cell viability. In HT-29 cells, inhibition of PKC (␣ and ␤ isoforms) activity augmented by twofold the cytotoxicity of both drugs. Induction of PKC by the phorbol ester phorbol myristate acetate (PMA) decreased the susceptibility of HT-29 cells to both TNF␣ and paclitaxel treatments. Resistance to apoptotic treatments was consistently observed in transfected HT-29 cells hyper-expressing PKC␤1. The present data implicate PKC (␣ and ␤) as a component of the mechanism responsible for the resistance of colon carcinoma cells to apoptotic drugs. MATERIAL AND METHODS Cells and chemicalsThe HT-29 human colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Rock...

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“…42,44 The PKC signaling pathway modulates cell resistance to apoptosis in a number of systems. 45,46 For example, the expression of PKC␤I confers resistance to tumor necrosis factor-␣ and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells. 45 In this regard, the activation of GABA receptors stimulates PKC activity in hippocampal and cultured spinal neurons.…”

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